Biomedical subjects

Hiroko K Kitamoto

Publications and source records attributed to Hiroko K Kitamoto.

4 recordsLinked to original sources

PubMed2005

Function of the cypX and moxY genes in aflatoxin biosynthesis in Aspergillus parasiticus.

The pathway oxoaverantin (OAVN) --> averufin (AVR) --> hydroxyversicolorone (HVN) --> versiconal hemiacetal acetate (VHA) is involved in aflatoxin biosynthesis, and the cypX and moxY genes, which are present in the aflatoxin gene cluster, have been previously suggested to be involved in this pathway. To clarify the function of these two genes in more detail, we disrupted the genes in aflatoxigenic Aspergillus parasiticus NRRL 2999. The cypX-deleted mutant lost aflatoxin productivity and accumulated AVR in the mycelia. Although this mutant converted HVN, versicolorone (VONE), VHA, and versiconol acetate (VOAc) to aflatoxins in feeding experiments, it could not produce aflatoxins from either OAVN or AVR. The moxY-deleted mutant also lost aflatoxin productivity, whereas it newly accumulated HVN and VONE. In feeding experiments, this mutant converted either VHA or VOAc to aflatoxins but did not convert OAVN, AVR, HVN, or VONE to aflatoxins. These results demonstrated that cypX encodes AVR monooxygenase, catalyzing the reaction from AVR to HVN, and moxY encodes HVN monooxygenase, catalyzing a Baeyer-Villiger reaction from HVN to VHA as well as from VONE to VOAc. In this work, we devised a simple and rapid method to extract DNA from many fungi for PCR analyses in which cell disruption with a shaker and phenol extraction were combined.

Aflatoxins↗

PubMed2005

Mannosyl-diinositolphospho-ceramide, the major yeast plasma membrane sphingolipid, governs toxicity of Kluyveromyces lactis zymocin.

Kluyveromyces lactis zymocin, a trimeric (alphabetagamma) protein toxin complex, inhibits proliferation of Saccharomyces cerevisiae cells. Here we present an analysis of kti6 mutants, which resist exogenous zymocin but are sensitive to intracellular expression of its inhibitory gamma-toxin subunit, suggesting that KTI6 encodes a factor needed for toxin entry into the cell. Consistent with altered cell surface properties, kti6 cells resist hygromycin B, syringomycin E, and nystatin, antibiotics that require intact membrane potentials or provoke membrane disruption. KTI6 is allelic to IPT1, coding for mannosyl-diinositolphospho-ceramide [M(IP)(2)C] synthase, which produces M(IP)(2)C, the major plasma membrane sphingolipid. kti6 membranes lack M(IP)(2)C and sphingolipid mutants that have reduced levels of M(IP)(2)C precursors, including the sphingolipid building block ceramide survive zymocin. In addition, kti6/ipt1 cells allow zymocin docking but prevent import of its toxic gamma-subunit. Genetic analysis indicates that Kti6 is likely to act upstream of lipid raft proton pump Kti10/Pma1, a previously identified zymocin sensitivity factor. In sum, M(IP)(2)C operates in a plasma membrane step that follows recognition of cell wall chitin by zymocin but precedes the involvement of elongator, the potential toxin target.

Alleles↗

PubMed2003

Functional characterization of two flap endonuclease-1 homologues in rice.

Flap endonuclease-1 (FEN-1) is an important enzyme involved in DNA replication and repair. Previously, we isolated and characterized a complementary DNA (cDNA) from rice (Oryza sativa) encoding a protein which shows homology with the eukaryotic flap endonuclease-1 (FEN-1). In this report, we found that rice (O. sativa L. cv. Nipponbare) possessed two FEN-1 homologues designated as OsFEN-1a and OsFEN-1b. The OsFEN-1a and OsFEN-1b genes were mapped to chromosome 5 and 3, respectively. Both genes contained 17 exons and 16 introns. Alignment of OsFEN-1a protein with OsFEN-1b protein showed a high degree of sequence similarity, particularly around the N and I domains. Northern hybridization and in situ hybridization analysis demonstrated preferential expression of OsFEN-1a and OsFEN-1b in proliferating tissues such as the shoot apical meristem or young leaves. The levels of OsFEN-1a and OsFEN-1b expression were significantly reduced when cell proliferation was temporarily halted by the removal of sucrose from the growth medium. When the growth-halted cells began to regrow following the addition of sucrose to the medium, both OsFEN-1a and OsFEN-1b were again expressed at high level. These results suggested that OsFEN-1a and OsFEN-1b are required for cell proliferation. Functional complementation assay suggested that OsFEN-1a cDNA had the ability to complement Saccharomyces cerevisiae rad27 null mutant. On the other hand, OsFEN-1b cDNA could not complement the rad27 mutant. The roles of OsFEN-1a and OsFEN-1b in plant DNA replication and repair are discussed.

Amino Acid Sequence↗

PubMed2003

Elongator's toxin-target (TOT) function is nuclear localization sequence dependent and suppressed by post-translational modification.

The toxin target (TOT) function of the Saccharomyces cerevisiae Elongator complex enables Kluyveromyces lactis zymocin to induce a G1 cell cycle arrest. Loss of a ubiquitin-related system (URM1-UBA4 ) and KTI11 enhances post-translational modification/proteolysis of Elongator subunit Tot1p (Elp1p) and abrogates its TOT function. Using TAP tagging, Kti11p contacts Elongator and translational proteins (Rps7Ap, Rps19Ap Eft2p, Yil103wp, Dph2p). Loss of YIL103w and DPH2 (involved in diphtheria toxicity) suppresses zymocicity implying that both toxins overlap in a manner mediated by Kti11p. Among the pool that co-fractionates with RNA polymerase II (pol II) and nucleolin, Nop1p, unmodified Tot1p dominates. Thus, modification/proteolysis may affect association of Elongator with pol II or its localization. Consistently, an Elongator-nuclear localization sequence (NLS) targets green fluorescent protein (GFP) to the nucleus, and its truncation yields TOT deficiency. Similarly, KAP120 deletion rescues cells from zymocin, suggesting that Elongator's TOT function requires NLS- and karyopherin-dependent nuclear import.

Active Transport, Cell Nucleus↗